Clinicopathologic and prognostic implications of HOXA gene and its associated long-noncoding RNAs expression in non-small cell carcinoma: A meta-analysis

Background: We conducted an investigation into the correlation between HOXA and associated long-noncoding RNAs, along with their clinicopathologic and prognostic features in non-small cell lung cancer (NSCLC). Methods: A comprehensive search across multiple electronic databases, including PubMed and the Web of Science, was conducted to identify relevant studies. The association between HOXA, clinicopathologic parameters, and prognosis was assessed using relative risk (RR) and hazard ratio (HR) with a 95% confidence interval (CI). Data compilation was performed using STATA 12.0 software. Results: A total of 11 trials involving 2058 patients with NSCLC were included in our study. Significant correlations were observed between HOXA-AS2 and TNM stage (III-IV) (RR=2.173, 95% CI: 1.386–5.437, P< 0.05) and HOTTIP and age (≥60-year-old) (RR=2.628, 95% CI: 1.185–5.829, P< 0.05) and non-smoking (RR=0.387, 95% CI: 0.156–0.959, P< 0.05). The combined results indicated a significant association between HOXA5 and increased overall survival (HR = 0.69, 95% CI = 0.57–0.84, P < .001). Additionally, HOXA-AS2, HOXA11 and HOTTIP were identified as potential independent predictors for poorer OS (HOXA-AS2: HR =3.48, 95% CI = 1.95 to 6.21, P < 0.05; HOXA11: HR=1.39, 95%CI = 1.08 to 1.79, P < 0.05; HOTTIP: HR=2.44, 95%CI = 1.10 to 5.42, P < 0.05). The prognostic significance of HOXA1, HOXA3 and HOXA4 was uncertain (HOXA1: HR=1.40, 95% CI =0.28 to 7.06, P > 0.05; HOXA3: HR=1.20, 95% CI = 0.96 to 1.50, P > 0.05; HOXA4: HR=0.97, 95% CI = 0.77 to 1.23, P > 0.05). Conclusions: The HOXA gene family has some potential to emerge as a novel prognostic factor for NSCLC and is correlated with some clinicopathological parameters such as the TNM stage, age and smoking. However, further meticulously designed prospective studies are warranted to substantiate these findings.


Introduction
Non-small cell lung cancer (NSCLC) constitutes 85% of all primary pulmonary carcinomas and stands as the leading cause of cancer-related mortality worldwide. [1,2]Lung cancer, characterized by a complex biological process, arises from the intricate dysregulation of various oncogenes linked to cancer development. [3]Despite advancements in multimodal therapies involving surgery, radiation, and chemotherapy for NSCLC, the 5-year overall survival (OS) rate remains suboptimal. [4]Consequently, the quest for precise and specific indicators to enhance the survival rate in NSCLC remains an imperative undertaking.
Homeobox (HOX) plays a crucial role in embryonic growth and tumor development.Among the 39 HOX genes, HOXA, HOXB, HOXC, and HOXD are located on different chromosomes (7p15.2,17, 21, 12, 13, 12, 13, and 2, 3.1). [5]The human HOX gene clusters encompass nonprotein-encoding genes, generating numerous long noncoding RNAs (lncRNAs) and evolutionarily conserved microRNAs. [6,7]The nonprotein-encoding RNAs within the HOX clusters play a pivotal role in cancer development by regulating the HOX genes. [8]tudies have indicated a potential correlation between the expression of HOXA genes in NSCLC and tumor invasiveness, patient prognosis, and treatment response.Due to the involvement of HOXA genes in cell differentiation and tumor progression, they emerge as promising targets for future NSCLC therapies.The development of drugs specifically addressing these genes holds the promise of offering more efficacious treatments for NSCLC patients.In-depth exploration of the precise mechanism of action of HOXA genes in NSCLC development can contribute to unveiling the pathogenesis of NSCLC, thereby fostering the formulation of more effective prevention and treatment strategies.Understanding the intricate relationship between HOXA genes and NSCLC establishes a robust foundation for personalized medicine.By scrutinizing the expression of HOXA genes in a patient's tumor, it may be feasible to tailor treatment plans that align more closely with the patient's individual conditions.
Thus, we conducted meta-analyses on HOXA and relevant lncRNAs in NSCLC to comprehensively assess their clinical significance, prognostic implications, and therapeutic potential.This rigorous examination aims to unravel the intricate expression patterns of the HOXA gene family and its associated lncRNAs in NSCLC through a meta-analytical approach.Our study is not solely focused on elucidating the fundamental scientific contributions of these molecules to tumor development but, more crucially, on accentuating their significant clinical relevance in advancing the diagnosis, treatment, and prognosis evaluation of NSCLC.Looking forward, the ongoing commitment to expanding our knowledge in this domain is imperative.This dedication plays a pivotal role in advancing therapeutic efficacy and markedly enhancing the quality of life for individuals affected by NSCLC.

Materials and methods
The present meta-analyses adhere to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. [9]1.Document retrieval strategy An extensive and systematic search was conducted in 2 electronic databases, namely PubMed and Web of Science, covering the period from November 2014 to May 19, 2023.The search terms "Homeobox A," "Hoxa," "Cancer, Non-small Cell Lung," and "NSCLC" were utilized.Both MeSH terms and free-text phrases were employed to maximize sensitivity.

Criteria for inclusion and exclusion
Classification criteria: (1) NSCLC confirmation through pathology.( 2) Subjects classified into HOXA-positive and HOXAnegative based on HOXA expression, and their prognosis compared.(3) Adequate data availability for calculating hazard ratio (HR) with 95% confidence interval (CI) for beneficial outcomes, even if not explicitly mentioned in the essays.
Exclusion criteria: (1) exclusion of letters, reviews, animal studies, case reports, and conference abstracts.(2) In cases of data duplication or overlap, only the most recent publications were considered.Document retrieval and selection: 2 independent researchers, Qi Wang and Jinyang Huang, conducted the document retrieval and selection.Any disagreements were resolved through group negotiations until a consensus was reached (Fig. 1).

Extracting data
For each of the mentioned studies, all data have been obtained separately by the 2 authors (Qi Wang and Jinyang Huang): the title of the primary author, the date of release, the country in which the trial was carried out, the size of the sample, HOXApositive, the tumor-node-metastasis (TNM) phase, and HR with 95% CI for the respective benefit outcomes.

Evaluation of quality
All included studies underwent assessment using the Newcastle-Ottawa Quality Rating Scale (NOS). [10]Studies with a score of 6 or more were considered qualitative.In case-control studies, the NOS scale primarily evaluates 3 aspects: (1) selection of study population, which assesses the representativeness of study subjects and the appropriateness of selecting study and control groups, covering 4 items: definition of the study population, source of study subjects, selection of the control group, and determination of exposure.(2) Assessment of exposure and outcome, focusing on accurately and comprehensively evaluating the exposure and outcome of interest in the study, comprising 2 items: assessment of exposure and assessment of outcome.
(3) Comparability of outcomes, which mainly assesses the comparability of outcomes between the study and control groups, including 2 items: analysis of outcomes and interpretation of outcomes.

Analysis of statistics
All statistical analyses were conducted using STATA 12.0.The association between HOXA and clinicopathologic features was assessed through a combined relative risk with a 95% CI, and a combined HR analysis of HOXA and outcomes was performed with a 95% CI.Whenever possible, HR with 95% CI values from multivariate models were utilized.In cases where these values were not directly reported, estimates were derived based on Kaplan-Meier data using the methodology described by Tierney et al. [11] The Chi-square Q test was employed to evaluate the degree of variation among study results.The Q statistic efficiently quantifies heterogeneity by comparing observed and expected variations through chi-square tests.Calculating Q values enabled the identification of statistically significant differences between the results of individual studies.To comprehensively assess the extent and source of heterogeneity, the I² statistical method was further employed.I², a relative measure representing the proportional variation in effect size, serves as a quantitative indicator of heterogeneity degree, ranging from 0% to 100%, with higher I² values indicating greater heterogeneity. [12]A random-effect model was applied in the presence of marked variability (P < .10and/or I² > 50%); otherwise, a fixed-effect model was applied. [13]Subsequently, a subgroup analysis was conducted, stratified by the source of HR, revealing that the source of HR might influence the prognostic value of the HOXA5 gene in NSCLC.The significant association of HOXA5 with OS was observed only in reported sources.[16] A log-rank P-value of < 0.05 was considered significant.

Key features of the covered studies
The involved trials were all retrospective with a NOS rating of 6 or above.Among the 11 trials included, the majority (10/11) were conducted in China.The percentage of subjects exhibiting HOXA expression ranged from 29.4% to 65.3%, and the sample sizes varied between 10 and 719.Furthermore, the HOXA expression state was assessed through immunohistochemistry in all the trials included.More detailed features are presented in Table 1.

Publication bias
We conducted Begg's Funnel Plot and Egger's Test to assess potential publication bias.In the absence of bias, these points should be symmetrically distributed on both sides of the funnel.Asymmetries or missing points may indicate publication bias.A significant slope in the regression line suggests an abnormal association between the effect size and standard error, indicating publication bias.The Begg's funnel plot exhibits symmetry, and Egger's test yields a P-value of 0.399 (overall efficacy) (Figure 4).

Discussion
To date, research has demonstrated the crucial involvement of HOXA and associated lncRNAs in the development and advancement of various cancer types, including breast cancer and NSCLC.Our study posits that the HOXA5 gene may function as a tumor suppressor in NSCLC.HOX transcription factors play a crucial role in regulating signaling pathways that control organ morphogenesis and maintain cell fate and differentiation in adults. [14,16]Recent research has delved into the exploration of Hox genes' involvement in organogenesis, with a specific focus on the lung. [28,29] Te underlying mechanism of HOXA5 in lung growth appears to be modulated by Notch signaling. [30][33] Notably, methylation of the HOXA5 promoter is detected in pulmonary carcinoma, [34] with approximately 81.3% of NSCLC cases exhibiting methylated HOXA5 promoter.Additionally, RT-PCR and immunohistochemistry have revealed a correlation between HOXA5 methylation and gene expression. [35]Our findings thus far indicate higher expression levels of HOXA5 in lessaggressive cells compared to their more aggressive counterparts.Furthermore, the overexpression of HOXA5 has been shown to suppress the invasion capacity of pulmonary carcinoma cells. [36]Consequently, we posit that HOXA5 may serve as a potent inhibitor of NSCLC.
Recently, evidence has emerged suggesting a collaborative role of HOXA5 and p53 in suppressing the invasion of NSCLC.Immunohistochemistry assays for p53 and HOXA5 Meta-analyses for the association of HOXA5 expression with survival of non-small cell lung cancer.

Table 4
The association of HOXA-AS2, HOXA11,HOTTIP, HOXA1, HOXA3 and HOXA4 expression with survival of non-small-cell lung cancer.conducted on primary NSCLC samples revealed that patients exhibiting no immune response to either p53 or HOXA5 experienced unfavorable outcomes. [37]Notably, the overexpression of HOXA5 in lung adenocarcinoma cells demonstrated a restraining effect on proliferation, invasion, and filopodia proliferation, along with the inhibition of in vivo metastasis.Conversely, the knockout of HOXA5 was associated with increased invasion of pulmonary carcinoma cells. [36]pigenetic regulation has been identified as a key factor governing HOX expression, with methylation playing a pivotal role in HOX silencing.40] The Hox transcript antisense intergenic RNA (HOTTIP) is situated on the 5' side of the HOXA group and is transcribed as a functional lncRNA.[43][44][45][46][47][48][49][50][51] In small cell lung cancer (SCLC), HOTTIP expression has been correlated with clinical staging, survival rates, and responses to chemotherapy. [52,53] hrough the modulation of the miR-574-5p/EZH1 axis, overexpression of HOTTIP has been shown to enhance SCLC cell proliferation and cell cycle progression. [53]Moreover, HOTTIP regulates the apoptotic activity and chemoresistance of SCLC cells by interacting with miR-216a, thereby increasing BCL-2 expression and inhibiting apoptosis. [52]In addition to its role in SCLC, HOTTIP exhibits elevated expression in NSCLC, where its overexpression promotes cell proliferation and migration.Notably, HOTTIP overexpression also negatively regulates HOXA13. [54]urthermore, the overexpression of HOTTIP contributes to drug resistance in lung adenocarcinoma through the modulation of AKT signaling pathways. [55]Collectively, these findings suggest that HOTTIP, as a lncRNA, serves as a crucial marker in the development of pulmonary carcinoma.

Author
In the context of NSCLC, HOXA genes play crucial roles in multiple biological processes relevant to tumor development and progression.Firstly, HOXA genes exert regulatory control over cell proliferation and apoptosis, pivotal processes in tumor growth and maintenance.Certain HOXA genes have demonstrated associations with apoptosis inhibition and the promotion of cell proliferation in NSCLC cells.Secondly, HOXA genes influence the tumor microenvironment by modulating the expression of genes involved in angiogenesis, invasion, and metastasis.This modulation significantly impacts the ability of NSCLC cells to disseminate and form secondary tumors.Thirdly, HOXA genes serve as targets for epigenetic regulation, including DNA methylation and histone modification, leading to altered expression in cancer cells.Epigenetic silencing of specific HOXA genes has been reported in NSCLC, contributing to tumor progression.Fourth, HOXA genes participate in various signaling pathways crucial for NSCLC development, such as the Wnt signaling pathway.The interplay between HOXA genes and these pathways influences tumor cell behavior and responses to therapeutic interventions.Fifth, HOXA genes are implicated in the regulation of cancer stem cells, believed to be responsible for tumor recurrence and treatment resistance.Certain HOXA genes' expression is linked to the maintenance of stem cell-like properties in NSCLC cells.
In terms of clinical significance, the aberrant expression of HOXA genes may function as a molecular marker for NSCLC, facilitating the identification of tumors with invasive growth potential.Elevated expression of HOXA genes correlates with poor prognosis in NSCLC patients, making it a potential biomarker for prognosis assessment.Given the pivotal role of HOXA genes in tumor development, they emerge as potential therapeutic targets for NSCLC, particularly in patients lacking conventional treatment targets.Future research avenues can be delineated as follows: delve deeper into the specific mechanisms of HOXA genes in NSCLC, exploring their impact on the tumor microenvironment and interactions with other molecular pathways; employ gene-editing techniques like CRISPR/Cas9 to precisely edit HOXA genes and unravel their function in tumor development; assess the safety and efficacy of targeted therapies against HOXA genes in animal models; conduct clinical trials for NSCLC patients with HOXA gene mutations to evaluate the clinical benefits of targeted therapies; explore the synergistic application of HOXA gene inhibitors with other treatment modalities, such as immunotherapy, to enhance therapeutic effectiveness.Real-world applications include offering personalized treatment recommendations based on the expression status of HOXA genes, selecting the most appropriate treatment strategy; developing inhibitors targeting HOXA genes to introduce novel treatment options for NSCLC patients; monitoring disease progression and therapeutic efficacy through regular detection of HOXA gene expression levels; integrating HOXA gene testing into the molecular diagnostic panel for NSCLC, aligning with the goals of precision medicine.
It is concluded that HOXA genes, along with other lncRNAs, show promise in predicting the outcome of pulmonary carcinoma.Moreover, additional research is warranted to ascertain whether HOXA and related lncRNAs might exert a substantial impact on the prognosis in different subtypes of NSCLC.

Limitations
The meta-analysis is subject to several limitations.Firstly, all included studies are retrospective with relatively small sample sizes, introducing a potential bias.Secondly, due to the absence of stratified data in the original studies, we were unable to conduct subgroup analyses based on age, sex, TNM, or other prognostic factors.Thirdly, while our results did not indicate apparent bias, there may be inherent bias as we only considered English-language publications.Fourthly, a majority of the studies originated from Asia, introducing a potential regional bias.Therefore, it is imperative to conduct additional research in non-Asian populations to validate the findings of the meta-analyses.

Conclusion
Hence, the HOXA gene family has some potential to serve as a novel prognostic factor for NSCLC and is correlated with some clinicopathological parameters including the TNM stage, age and smoking.However, substantiating our results would necessitate a larger number of prospective trials.

Figure 1 .
Figure 1.Flow diagram of the literature review.

Figure 2 .
Figure 2. Forest plot of the association between HOXA gene and its associated long-noncoding RNAs and overall survival.

Figure 3 .
Figure 3. Forest plot of the association between HOXA5 gene and overall survival.

Figure 4 .
Figure 4. Begg's funnel plot of the association between HOXA gene and its associated long non-coding RNAs and overall survival.

Table 1
Basic characteristics of included studies.

Table 2
Associations of HOXA expression with clinicopathological characteristics in non-small cell lung cancer.